Page 316 - Haematologica March 2020

P. Ferraresi et al.
mon variants have been identified in the F7 gene (http://f7-db.eahad.org/). Nevertheless, in spite of an exhaustive direct sequencing of F7 exons and exon-intron junctions and of the proximal promoter region, a signifi- cant proportion of defective alleles has still not been iden- tified. The rate of uncharacterized F7 disease alleles ranges from 2% to 8%7-10 in Europe, and a similar esti- mate (7%) was made in India.11
In this context, subtle intronic variations outside the routinely sequenced exon-intron boundaries could have a pathological impact by impairing the splicing process. In fact, precise exon definition during RNA processing requires the interplay among several exonic and intronic splicing regulatory elements,12 which can be altered by nucleotide changes and lead to aberrant splicing.4,13-17 Various examples of “deep” intronic changes associated with mis-splicing have been reported in human disorders, including those involved in coagulation.18-20 It is worth noting that RNA splicing can be modulated for different purposes, including the development of new thera- pies.14,20-26 In this context, next-generation sequencing (NGS) could represent a powerful tool to characterize gene defects in patients with unknown alleles; however, only a few studies have been conducted in coagulation factor disorders.27-29
Here, we investigated 13 patients with FVII deficiency forms that have not been explained by mutations identi- fied by conventional sequencing and used NGS to identi- fy six rare intronic variations that could be causative. Through expression studies, we demonstrated that two of them lead to aberrant splicing, which explained the residual FVII levels in most patients and, intriguingly, that these can be rescued by an antisense-based correction approach.
Table 1. Features of the investigated FVII deficient patients.
Methods
Patients
Since 1997, 400 FVII-deficient patients with FVII coagulant activity (FVII:C) levels <30% were referred to our laboratory for genetic analysis through conventional screening. Among them, 13 (Table 1) showed a genetic profile that, considering the iden- tified mutated alleles10,30-32 and the major F7 functional polymor- phisms (c.-325_-324insCCTATATCCT, A2 allele; p.Arg413Gln change, M2 allele),33-37 appeared to be incompatible with the reduced FVII levels. The local Institutional Review Board approved the study and patients provided informed consent.
Measurement of FVII levels
FVII:C and FVII antigen levels were determined by the one- stage method38 and Enzyme-Linked-Immunoabsorbant-Assay (Diagnostica Stago, Asnière sur Seine, France), respectively.
DNA genotyping and next-generation sequencing
Conventional Sanger technology was exploited to sequence the F7 exons, including the intronic boundaries and the 5’ untranslated region. Large rearrangements were ruled out using semi-quantitative multiplex fluorescent-polymerase chain reac- tion (SQF-PCR) assays.39 NGS of the F7 gene was designed to cover the intronic regions with the exception of the highly repet- itive GC-rich region in intron 2 (legacy nomenclature, intron 1b). The probe-capture custom design targeted the 14893-pbF7 gene in two parts: chr13:113759000-113761350 and chr13:113764600-113775100 accounting for a total of 12,850 base-pairs with a gap of 3250 bp. DNA library generation was performed using the Custom SureSelectQXT Target Enrichment system (Agilent, Santa Clara, CA, USA) on a MiSeq platform (Illumina, San Diego, CA, USA). The sequencing data were stored in FASTQ format and analyzed using two bio-informatics
Proband
#17
#19 #31
#113
#262
#341
#214 #28 #377 #284 * #90 * #15 #330
Origins
Maghreb countries
France France
France
France
France
France
France Maghreb countries Maghreb countries Lebanon Maghreb countries France
FVII:C (%)
3
FVII:Ag(%)
uk
Conventional sequencing
Polymorphic pattern c.325_324ins/ p.Arg413Gln
A1A1/M1M1
A1A2/M1M2 A1A1/M1M1
A1A1/M1M1
A1A1/M1M1
A1A1/M1M1
A1A1/M1M1 A1A1/M1M1 A2A2/M2M2 A1A2/M1M2 A1A1/M1M1 A1A1/M1M1 A2A2/M2M2
NGS sequencing
Mutation
p.Met358Ile10
p.Met1Val10 p.Cys162Tyr10
p.Arg364Trp30
p.Gln160Arg31
c.430+1G>A32
p.Arg58ProfsX92 p.Arg59Trp
Zygosity
het
het het
het
het
het
het het
Additional nucleotide change
Zygosity
2 <5 <1 19
1 66
3 uk
16 uk
1 3 13 39 23 uk 20 15 <1 uk 3 uk 10 uk
c.571+78G>A het
c.571+78G>A het
c.64+305G>A and het c.806-329G>A
c.571+78G>A and het
c.291+846C>T c.806-329G>A het
c.64+305G>A and het
c.681+132G>T c.571+78G>A het c.571+78G>A het c.572-392C>G het c.571+78G>A het c.571+78G>A hom c.571+78G>A hom c.572-392C>G hom
*Consanguinity. NGS: next-generation sequencing; uk: unknown; A1/A2: decanucleotide insertion c.-325_-324insCCTATATCCT promoter polymorphism rs5742910; M1/M2: p.Arg413Gln polymorphism: rs6046; FVII:C: FVII activity; FVII:Ag: FVII antigen; het: heterozygous; hom: homozygous.
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haematologica | 2020; 105(3)